High-resistance MDCK-C7 monolayers used for measuring invasive potency of tumour cells

Žák, Jan; Schneider, Stefan W.; Eue, Ines; Ludwig, Thomas; Oberleithner, Hans

doi:10.1007/s004240051037

Cited by 10 publications

(29 citation statements)

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“…1A). In the first set of experiments we investigated the invasive properties of all three cell lines with an electrophysiological cell-based invasion assay previously established in our laboratory 24,25 . One of the key features of this assay is the high sensitivity to evaluate very early steps of invasion 26 .…”

Section: Discussionmentioning

confidence: 99%

“…1A-C). To evaluate the progression of human malignant melanoma cells we applied a previously established electrophysiological invasion assay 24,25 .…”

Section: Knockdown Of Hpse Reduces Melanoma Cell Invasive Capacitymentioning

confidence: 99%

“…Transfections were performed as previously described 22 . TEER measurements were done with MDCK-C7 cells as reported earlier 24 .…”

Section: Cell Culturementioning

confidence: 99%

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Nuclear heparanase-1 activity suppresses melanoma progression via its DNA-binding affinity

et al. 2015

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Heparanase-1 (HPSE) plays a pivotal role in structural remodeling of the ECM and glycocalyx thus conferring protumorigenic, proangiogenic and prometastatic properties to many cancer entities. In addition to its extracellular function, recent studies suggest an intracellular activity of HPSE with a largely unknown significance during tumor progression. Therefore, we investigated the relevance of HPSE duality in malignant melanoma in vitro as well as in mouse melanoma models basing on the intradermal injection of transfected melanoma cells. In line with its extracellular action, HPSE-deficiency led to a reduced shedding of the glycocalyx accompanied by a reduced availability of VEGF affecting tumor growth and vascularization. In contrast, we measured an elevated expression of the protumorigenic factors pentraxin-3, tissue factor, TNF-and most prominently MMP-9 upon HPSE knockdown. In vivo, HPSE-deficiency was related to increased lymph node metastasis. While inhibition of its extracellular function heparin was unable to block the gene regulatory impact of HPSE we proposed an intracellular mechanism. Immunostainings revealed a counter-staining of HPSE and NF-B in the nucleus suggesting a close relationship between both proteins. This finding was further supported by the discovery of a direct charge-driven molecular interaction between HPSE and DNA by using atomic force microscopy and a co-precipitation approach. Our findings are novel and point towards a dual identity of HPSE in malignant melanoma with a protumorigenic extracellular activity and a tumor suppressive nuclear action. Identification of molecular strategies to shuttle extracellular HPSE into the nuclei of cancer cells envisions new therapeutic options. AbstractHeparanase-1 (HPSE) plays a pivotal role in structural remodeling of the ECM and glycocalyx thus conferring protumorigenic, proangiogenic and prometastatic properties to many cancer entities. In addition to its extracellular function, recent studies suggest an intracellular activity of HPSE with a largely unknown significance during tumor progression.Therefore, we investigated the relevance of HPSE duality in malignant melanoma in vitro as well as in mouse melanoma models basing on the intradermal injection of transfected melanoma cells. In line with its extracellular action, HPSE-deficiency led to a reduced shedding of the glycocalyx accompanied by a reduced availability of VEGF affecting tumor growth and vascularization. In contrast, we measured an elevated expression of the protumorigenic factors pentraxin-3, tissue factor, TNF-α and most prominently MMP-9 upon HPSE knockdown. In vivo, HPSE-deficiency was related to increased lymph node metastasis.While inhibition of its extracellular function heparin was unable to block the gene regulatory impact of HPSE we proposed an intracellular mechanism. Immunostainings revealed a counter-staining of HPSE and NF-κB in the nucleus suggesting a close relationship between both proteins. This finding was further supported by the discovery of a dir...

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Section: Discussionmentioning

confidence: 99%

“…1A-C). To evaluate the progression of human malignant melanoma cells we applied a previously established electrophysiological invasion assay 24,25 .…”

Section: Knockdown Of Hpse Reduces Melanoma Cell Invasive Capacitymentioning

confidence: 99%

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Nuclear heparanase-1 activity suppresses melanoma progression via its DNA-binding affinity

et al. 2015

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“…Highly malignant human amelanotic melanoma cells, subclone A7, are transfected with actin binding protein to maximize their migration activity (24). (25) were cultured at standard conditions as described previously (23).…”

Section: Methodsmentioning

confidence: 99%

“…Melanoma cells were cultured for 2 days as described previously (23). Cells inspected to be nearly confluent were washed three times and transferred to serum-free medium.…”

Section: Melanoma Cells and Madin-darby Canine Kidney Cells (Mdck)-c7mentioning

confidence: 99%

Microtubule-Dependent Matrix Metalloproteinase-2/Matrix Metalloproteinase-9 Exocytosis

et al. 2004

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Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that cleave and degrade a wide spectrum of extracellular matrix components. By enhancing turnover of extracellular matrix, MMP activity is also known to play a key role in tumor cell invasion. Because extracellular protease activity requires efficient release of these proteases to the cellular surface, we investigated storage, transport, and exocytosis of MMP-2 and MMP-9 in human melanoma cells using immunofluorescence, electrical, and biochemical techniques. Immunolabeling of melanoma cells with antibodies specific for MMP-2 and MMP-9 led to the identification of two distinct populations of small cytoplasmatic vesicles containing MMP-2 or MMP-9, respectively. In combination with ␣-tubulin-specific antibodies, both vesicle populations were found to be aligned along the microtubular network. Moreover, the molecular motor protein kinesin is shown to be localized on most of these vesicles, providing evidence that the identified vesicles are actively propelled along microtubules toward the plasma membrane. The functional relevance of these findings is demonstrated using low dosage (5.9 nmol/L) of paclitaxel to affect the microtubular function of melanoma cells. Although cell proliferation is not altered, paclitaxel treatment impairs secretion of MMP-2/MMP-9 and significantly reduces invasive activity in our new cell invasion assay. In conclusion, we demonstrate in melanoma cells that microtubule-dependent traffic of MMP-containing vesicles and exocytosis are critical steps for invasive behavior and therefore are potential targets for specific antitumor drugs.

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Local proteolytic activity in tumor cell invasion and metastasis

Ludwig¹

2005

BioEssays

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Proteolytic cleavage of extracellular matrix (ECM) is a critical regulator of many physiological and pathological events. It affects fundamental processes such as cell growth, differentiation, apoptosis and migration. Most proteases are produced as inactive proenzymes that undergo proteolytic cleavage for activation. Proteolytic activity is additionally modified by endogenous inhibitors. Mechanisms that localize and concentrate protease activity in the pericellular microenvironment of cells are prerequisites for processes like angiogenesis, bone development, inflammation and tumor cell invasion. Methods that enable real-time, high-resolution imaging and precise quantification of local proteolytic activity in vitro and in vivo remain major challenges. These methods will play an important role in the understanding of basic principles e.g. in cancer cell invasion, the identification of new therapeutical targets and hence drug design. This review highlights mechanisms and functions of local proteolytic activity with special emphasis on tumor cell invasion and metastasis, and focuses on techniques for the investigation of this process.

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High-resistance MDCK-C7 monolayers used for measuring invasive potency of tumour cells

Cited by 10 publications

References 0 publications

Nuclear heparanase-1 activity suppresses melanoma progression via its DNA-binding affinity

Nuclear heparanase-1 activity suppresses melanoma progression via its DNA-binding affinity

Microtubule-Dependent Matrix Metalloproteinase-2/Matrix Metalloproteinase-9 Exocytosis

Local proteolytic activity in tumor cell invasion and metastasis

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